1. Field of the Invention
The invention generally relates to improved diagnosis and treatment of mycobacterial infections. In particular, the invention provides methods for using spent culture supernatant, components of the spent culture supernatant, or cell extracts to enhance cultivation of mycobacteria, or to revive dormant mycobacteria bacilli.
2. Background of the Invention
Tuberculosis (TB) is a leading infectious killer worldwide with 8 million new cases and 2 million deaths a year (WHO Report on the Tuberculosis Epidemic, 2000). One third of the world population is latently infected with Mycobacterium tuberculosis. The success of M. tuberculosis as a human pathogen relates to its remarkable ability to persist for long periods of time in the face of immunity and chemotherapy.
The current TB treatment is suboptimal, requiring a minimum of 6 months using the WHO recommended treatment regimen (DOTS, Directly Observed Treatment, Shortcourse), which consists of 4 drugs isoniazid, rifampin, pyrazinamide and ethambutol used in combination (WHO Report on the Tuberculosis Epidemic, 2000). Such lengthy treatments are expensive, and are likely to be problematic in terms of patient compliance. In addition, one potentially catastrophic effect of the lengthy therapy is the development of drug-resistant TB.
This lengthy treatment is thought to be due to the presence of a population of dormant bacilli in vivo that are not effectively killed by current TB drugs (McKinney et al., 1998). Dormant bacilli can be demonstrated in the Cornell mouse model of dormancy (McCune et al, 1966), where mice infected with tubercle bacilli were treated with INH and PZA for 2 months, at which time no viable bacilli were demonstrable in the tissues as judged by colony forming units (CFU); yet disease relapsed with viable yet drug susceptible bacilli after cessation of treatment for 3 months in one third of mice or in almost all mice given immunosuppressing steroids. This suggests that the drugs are unable to eliminate dormant bacilli completely and that although the dormant bacilli do not form colonies on plates they are not dead and can revive and cause disease when the immune system is compromised.
The unresponsiveness of dormant or nongrowing bacilli to DOTS is phenotypic or physiologic but not genetic, so that when the dormant bacilli revive and start growing they become susceptible to TB drugs again. Therefore, agents that cause dormant bacilli to revive and resuscitate so that they respond to treatment are potential modulators of drug activity in the host and can be used for improved treatment of the disease by potentially shortening the treatment time.
The current diagnosis of tuberculosis still relies on culture of the M. tuberculosis organism as the definitive method of diagnosis. However, M. tuberculosis grows very slowly and it takes several weeks for the primary isolation of the bacilli from clinical specimens for confirmation of the disease. Current clinical diagnosis uses solid media such as Lowenstein-Jensen medium, 7H10 or 7H11 agar medium and liquid 7H12B medium as in BACTEC460 machine for primary isolation of the bacilli from clinical specimen. In general, the liquid 7H12B based medium is more sensitive in terms of primary isolation of positive cultures from clinical specimens. However, the current medium for isolation of M. tuberculosis from clinical specimens is not optimal. Even with liquid 7H12B medium in the presence of growth enhancing agent POES (polyoxyethelene stearate) (Becton Dickinson, Sparks, Md., U.S. Pat. No. 4,769,332), the isolation rate is about 80%, and some 20% samples which later prove to be containing the bacilli are not easily detected. Agents that can improve the primary isolation sensitivity and enhance the growth of M. tuberculosis should improve the ability to diagnose TB.
The present invention provides media and methods for enhancing the cultivation of mycobacteria, or reviving (resuscitating) dormant bacilli from mycobacterium species. The media and methods utilize mycobacterial products from the M. tuberculosis complex. By xe2x80x9cM. tuberculosis complexxe2x80x9d we mean M. tuberculosis complex organisms which include M. tuberculosis, Mycobacterium bovis, including the vaccine strain BCG, and Microbacterium microti. The products include cell extracts, early-stationary-phase culture supernatant (ESPSN), and stationary phase culture supernatant (SPSN), either crude or as substantially purified components from these sources. The products may be used to enhance the growth of mycobacterial species that are difficult to culture, and/or to effect the resuscitation of dormant mycobacteria bacilli.
It is an object of this invention to provide a supplemented medium for culturing mycobacterium species, for example Mycobacterium tuberculosis, Mycobacterium paratuberculosis, and Mycobacterium leprae. The medium comprises a cell extract from M. tuberculosis complex, or at least one product from M. tuberculosis complex (e.g. a component of early-stationary-phase culture supernatant (ESPSN), of stationary phase culture supernatant (SPSN), or of a cell extract) and a suitable culture medium. The cell extract or substantially purified product exhibits resuscitation activity for dormant bacilli of the mycobacterium species, or the ability to enhance the growth of the mycobacterium species. The substantially purified product may be a phospholipid or a component of a phospholipid, such as phosphotidyl-L-serine, dioleoyl phosphotidyl-L-serine, phosphotidylcholine, phosphotidylethanolamine, tuberculostearic acid, arachidonic acid, and fatty acids. Alternatively, the substantially purified product may be a protein or a fragment of a protein, e.g. protein Rv1147c (accession number F70875 in the National Institute of Health""s Entrez Protein Database), a peptide corresponding to SEQ ID NO. 1, a peptide corresponding to SEQ ID NO. 2, a peptide corresponding to SEQ ID NO. 3, a peptide corresponding to SEQ ID NO. 4 and a peptide corresponding to SEQ ID NO. 5. Suitable culture media to be supplemented include 7H12B, 7H9, 7H10, 7H11, Sauton""s medium, Dubos medium, and egg-based media (for example Lowenstein-Jensen medium). In addition, a mixture of substantially purified component of ESPSN or SPSN may be utilized, e.g. a mixture of phospholipids and/or proteins and peptides.
In addition, isolated and sterilized ESPSN or SPSN of M. tuberculosis complex can itself be utilized as a culture medium, or combined with a suitable fresh culture medium or other fresh nutrients to produce supplemented culture medium. Further, substantially purified components of ESPSN, SPSN, or mycobacterium cell extracts that exhibit resuscitation activity for dormant bacilli of the mycobacterium species may be added.
In another aspect, the present invention provides a method for reviving dormant mycobacterium bacilli of, for example Mycobacterium tuberculosis, Mycobacterium paratuberculosis, and Mycobacterium leprae. According to the method, dormant bacilli are exposed to a cell extract or at least one substantially purified product of M. tuberculosis complex (e.g. a component of early-stationary-phase culture supernatant (ESPSN), of stationary phase culture supernatant (SPSN), or of a cell extract). The cell extract and substantially purified product exhibit resuscitation activity for dormant bacilli of the mycobacterium species, and the cell extract or product is present in sufficient quantity to effect revival of the dormant bacilli. The substantially purified product may be a phospholipid or component thereof, e.g. phosphotidyl-L-serine, dioleoyl phosphotidyl-L-serine, phosphotidylcholine, or phosphotidylethanolamine, tuberculostearic acid, arachidonic acid, and fatty acids. Alternatively, the substantially purified product may be a protein or a fragment of a protein, e.g., protein Rv1147c, a peptide corresponding to SEQ ID NO. 1, a peptide corresponding to SEQ ID NO. 2, a peptide corresponding to SEQ ID NO. 3, a peptide corresponding to SEQ ID NO. 4 and a peptide corresponding to SEQ ID NO. 5. In addition, a mixture of substantially purified products may be utilized, e.g. a mixture of phospholipids and/or proteins and peptides.
In addition, dormant bacilli may be revived by exposure to cell extract or isolated and sterilized ESPSN or SPSN of M. tuberculosis complex, which may also be combined with a suitable fresh culture medium or other fresh nutrients (e.g. substantially purified components of ESPSN, SPSN, or mycobacterium cell extracts that exhibit resuscitation activity for dormant bacilli).
In yet another aspect of the present invention, a method for the diagnosis of an infection caused by a mycobacterium species (for example Mycobacterium tuberculosis, Mycobacterium paratuberculosis, and Mycobacterium leprae) is provided. The method comprises combining a sample for which the presence or absence of the mycobacterium species is to be determined with medium supplemented with cell extract or at least one substantially purified product M. tuberculosis complex (e.g. a component of early-stationary-phase culture supernatant (ESPSN), of stationary phase culture supernatant (SPSN), or of a cell extract); and analyzing the culture for the presence of the mycobacterium species. The cell extract and substantially purified product exhibits growth enhancement and/or resuscitation activity for dormant bacilli of the mycobacterium species. If the mycobacterium species is found in the culture, this indicates a positive diagnosis for the infection. The substantially purified product may be a phospholipid or component of a phospholipid, e.g. phosphotidyl-L-serine, dioleoyl phosphotidyl-L-serine, phosphotidylcholine, or phosphotidylethanolamine, tuberculostearic acid, arachidonic acid, and fatty acids. Alternatively, the substantially purified product may be a protein or a fragment of a protein, e.g. protein Rv1147c, a peptide corresponding to SEQ ID NO. 1, a peptide corresponding to SEQ ID NO. 2, a peptide corresponding to SEQ ID NO. 3, a peptide corresponding to SEQ ID NO. 4 and a peptide corresponding to SEQ ID NO. 5. In addition, a mixture of substantially purified products may be utilized, e.g. a mixture of phospholipids and/or proteins and peptides.
In addition, the method for the diagnosis of an infection caused by a mycobacterium species may comprise combining a sample for which the presence or absence of the mycobacterium species is to be determined with isolated and sterilized ESPSN or SPSN of M. tuberculosis complex, which may also be combined with a suitable fresh culture medium or other fresh nutrients (e.g. substantially purified components of ESPSN, SPSN, or mycobacterium cell extracts that growth enhancing or resuscitation activity for dormant bacilli).
In another aspect of the present invention, a kit for the diagnosis of an infection caused by a mycobacterium species (for example Mycobacterium tuberculosis, Mycobacterium paratuberculosis, and Mycobacterium leprae) is provided. The kit includes a sealed container of medium supplemented with cell extract or at least one substantially purified product of M. tuberculosis complex (e.g. a component of early-stationary-phase culture supernatant (ESPSN), of stationary phase culture supernatant (SPSN), or of a cell extract), which may further comprise additional fresh media or nutrients. The cell extract or substantially purified product exhibits growth enhancement and/or resuscitation activity for dormant bacilli of the mycobacterium species. The substantially purified product may be a phospholipid or a component of a phospholipid e.g. phosphotidyl-L-serine, dioleoyl phosphotidyl-L-serine, phosphotidylcholine, or phosphotidylethanolamine, tuberculostearic acid, arachidonic acid, and fatty acids. Alternatively, the substantially purified product may be a protein or a fragment of a protein, e.g. protein Rv1147c, a peptide corresponding to SEQ ID NO. 1, a peptide corresponding to SEQ ID NO. 2, a peptide corresponding to SEQ ID NO. 3, a peptide corresponding to SEQ ID NO. 4 and a peptide corresponding to SEQ ID NO. 5. In addition, a mixture of substantially purified products may be utilized, e.g. a mixture of phospholipids and/or proteins and peptides.
In addition, the kit may comprise isolated and sterilized ESPSN or SPSN of M. tuberculosis complex, which may also be combined with a suitable fresh culture medium or other fresh nutrients (e.g. substantially purified components of ESPSN, SPSN, or mycobacterium cell extracts that exhibit growth enhancement or resuscitation activity for dormant bacilli).
The present invention further provides a method for the treatment of an infection caused by a mycobacterium species (for example Mycobacterium tuberculosis, Mycobacterium paratuberculosis, and Mycobacterium leprae). According to the method, cell extract or at least one substantially purified product of M. tuberculosis complex (e.g. a component of early-stationary-phase culture supernatant (ESPSN), of stationary phase culture supernatant (SPSN), or of a cell extract), is administered to said patient, in conjunction with drugs of an established treatment protocol for the infection in order to ameliorate symptoms associated with the infection. Administering such a substance results in the revival of dormant bacilli of the mycobacterium species in the patient, thus making the bacilli susceptible to treatment with an antibiotic. (Dormant bacilli are otherwise not susceptible to current drug therapy protocols.) The substantially purified product may be a phospholipid e.g. phosphotidyl-L-serine, dioleoyl phosphotidyl-L-serine, phosphotidylcholine, or phosphotidylethanolamine, tuberculostearic acid, arachidonic acid, and fatty acids. Alternatively, the substantially purified product may be a protein or a fragment of a protein, e.g. protein Rv1147c, a peptide corresponding to SEQ ID NO. 1, a peptide corresponding to SEQ ID NO. 2, a peptide corresponding to SEQ ID NO. 3, a peptide corresponding to SEQ ID NO. 4 and a peptide corresponding to SEQ ID NO. 5. In addition, a mixture of substantially purified products may be utilized, e.g. a mixture of phospholipids and/or proteins and peptides.
In another aspect, the present invention provides a pharmacological agent for the treatment of an infection caused by a mycobacterium species (for example Mycobacterium tuberculosis, Mycobacterium paratuberculosis, and Mycobacterium leprae). The agent comprises cell extract or at least one substantially purified product of M. tuberculosis complex (e.g. a component of early-stationary-phase culture supernatant (ESPSN), of stationary phase culture supernatant (SPSN), or of a cell extract). The substantially purified product may be a phospholipid, e.g. phosphotidyl-L-serine, dioleoyl phosphotidyl-L-serine, phosphotidylcholine, or phosphotidylethanolamine, tuberculostearic acid, arachidonic acid, and fatty acids. Alternatively, the substantially purified product may be a protein or a fragment of a protein, (for Mycobacterium tuberculosis, protein Rv1147c, a peptide corresponding to SEQ ID NO. 1, a peptide corresponding to SEQ ID NO. 2, a peptide corresponding to SEQ ID NO. 3, a peptide corresponding to SEQ ID NO. 4 and a peptide corresponding to SEQ ID NO. 5., and a physiologically suitable carrier. In addition, a mixture of substantially purified products may be utilized, e.g. a mixture of phospholipids and/or proteins and peptides.
The present invention provides media and methods for enhancing the cultivation of mycobacteria, or reviving (resuscitating) dormant bacilli from mycobacterium species. The media and methods utilize mycobacterial products from, for example, the early stationary phase supernatant (ESPSN), the stationary phase supernatant (SPSN), or cell extracts of cultures of mycobacterium. The products exhibit growth enhancement of mycobacteria, or resuscitation activity for dormant bacilli of mycobacterium species, examples of which include but are not limited to Mycobacterium tuberculosis, Mycobacterium paratuberculosis, and Mycobacterium leprae. By the xe2x80x9crevivalxe2x80x9d or xe2x80x9cresuscitationxe2x80x9d of dormant bacilli, we mean that bacilli that do not display characteristics of growth (e.g. they do not form colonies on suitable media, or do not test as live with FDA-EB staining) obtain the ability to display growth-related characteristics, (e.g. the ability to form colonies on suitable media or to test as live with FDA-EB staining). By xe2x80x9cenhancement of growthxe2x80x9d we mean that the amount of growth exhibited by a mycobacterial culture is increased at least about two-fold compared to a control culture to which the supplement has not been added.
The invention is predicated on the novel finding that the early stationary phase supernatant (ESPSN) and the stationary phase supernatant (SPSN) of cultures of the mycobacterium have the ability to cause the revival or resuscitation of dormant bacilli, and to generally enhance the growth of mycobacterial cultures.
When a batch culture of bacteria grows to stationary phase, bacterial growth halts because of the exhaustion of essential nutrients and accumulation of toxic products (Postgate, 1967). Upon extended incubation in stationary phase, bacteria begin to die and viability of the culture decreases. The number of colony forming units (CFU) of an aged stationary batch culture is often orders of magnitude less than the total number of bacteria in the culture (Amann et al., 1995; Kaprelyants and Kell, 1993; Votyakova et al., 1994). The nonculturable bacterial population consists of dead cells as well as injured or dormant cells. The present invention capitalizes on the discovery that dormant M. tuberculosis cells can be induced to revive upon exposure to ESPSN or SPSN. Further, individual components of the ESPSN and SPSN that possess this ability have been identified and substantially purified. Several embodiments of this discovery are herein disclosed, namely media for culturing mycobacterium, methods for reviving dormant mycobacterium bacilli, improved methods for the diagnosis of infections caused by mycobacterium, kits for the diagnosis of infections caused by mycobacterium, improved methods for the treatment of infections caused by mycobacterium, and a pharmaceutical preparation for the treatment of infections caused by mycobacterium.
The present invention provides media for culturing a mycobacterium species comprising the isolated and sterilized ESPSN or SPSN of the mycobacterium species. In a preferred embodiment of the present invention, the mycobacterium species is Mycobacterium tuberculosis (M. tuberculosis). Those of skill in the art will recognize, however, that the compositions and methods disclosed herein are equally applicable to other mycobacterial species, including but not limited to Mycobacterium paratuberculosis (the causative agent of Crohn""s disease in humans and Johne""s disease in cattle), and Mycobacterium leprae (the causative agent of leprosy). Mycobacterial species have many commonalities, such as the difficulty of establishing them in culture. It is well known that to initiate the growth of M. tuberculosis, a large inocula are needed and small inocula often fail to initiate the growth. It is very likely that growth of M. tuberculosis requires autocrine factors secreted by adjacent bacilli for the small inocula to grow. Supplement of such factors from ESPSN or SPSN or cell extract containing the autocrine factors to the culture media can potentially allow small inocula to grow, which otherwise cannot grow. Thus this method of cultivation using ESPSN, or SPSN, or cell extract of M. tuberculosis complex organisms can be used to improve the sensitivity of isolation of M. tuberculosis complex from clinical specimens, and also for improved growth of the M. tuberculosis complex organisms for research lab use. Therefore, while the methodology described herein utilizes M. tuberculosis in many examples, one of skill in the art will recognize that it can readily be adapted to other mycobacterial species.
By ESPSN we mean the supernatant obtained from the early stationary phase of a culture, e.g. for M. tuberculosis complex a supernatant obtained from an approximately 3-4 week old culture. By SPSN we mean the supernatant obtained from the stationary phase, e.g. for M. tuberculosis complex from an approximately 1-2 month old culture. By xe2x80x9cisolated and sterilizedxe2x80x9d we mean that the supernatant has been treated to remove particulate matter and sterilized to eliminate mycobacteria or other contaminating organisms. Collection of the supernatant, removal of particulate matter, and sterilization may be accomplished by any of a variety of means which are well known to those of skill in the art. For example, collection may be accomplished via centrifugation (e.g. about 6,000xc3x97 g for about 20 minutes) followed by sterilization via filtration, e.g. through a 0.22 xcexcm filter. Those of skill in the art will recognize that other suitable means of accomplishing collection and sterilization of the supernatant are available and well-known, including but not limited to centrifugation followed by UV irradiation.
As demonstrated herein, the isolated ESPSN and SPSN from M. tuberculosis complex contain factors which promote the revival or resuscitation of dormant mycobacterium bacilli. In order to be utilized for this purpose, the isolated ESPSN or SPSN may be used alone, or may be supplemented or enriched with various nutrients in order to enhance growth of a new inoculum. Those of skill in the art will recognize that media comprised of ESPSN or SPSN may be enriched by the addition of other substances known to be conducive to the growth of bacteria in general, and of mycobacterium in particular, for example, Tween 80, albumin-dextrose-catalase, various salts and nutrients, buffering agents, fatty acids and the like. Nutrients may be added individually to the ESPSN or SPSN. Alternatively, the ESPSN or SPSN may be combined with other fresh media to supply the nutrients.
In order to obtain ESPSN, SPSN or cell extracts, a culture of mycobacterium must be established. Methods of culturing mycobacterium are well-known to those of skill in the art (see, for example Kent, P T, Kubica, G P. Public health mycobacteriology. A guide for the level III laboratory. Atlanta, Ga.; Centers for Disease Control, 1985; Nolte, F S, Metchcock B. Mycobacterium. In: Murray P R, Baron E J, Pfaller M A, Tenowver F C, Yolken R H ed. Manual of clinical microbiology, 6th ed. Washington D.C.; ASM Press 1995: pp 400-437) as are methods of obtaining a suitable supernatant from such a culture. In general, an inoculum from an appropriate strain of mycobacterium is introduced into a culture medium and allowed to grow for the requisite period of time under conditions that are well-known to those of skill in the art (e.g. sterile conditions, about 37xc2x0 C., with or without agitation of the culture). Suitable strains of mycobacterium which may be utilized in the practice of the present invention include but are not limited to M. tuberculosis H37Ra, [M. fortuitum] M. fortuitum, etc. Useful media for culturing M. tuberculosis strains include but are not limited to 7H9, 7H10, 7H11, 7H12B, Sauton""s medium, Dubos medium, egg-based media such as Lowenstein-Jensen medium, and the like, which are readily commercially available (e.g. Difco). Further, those of skill in the art will recognize that such media may be supplemented with substances such as Tween 80 (0.05%), albumin-dextrose-catalase (ADC), fatty acids, and the like.
The inoculated cultures are grown under suitable conditions until early stationary phase (ESP) is achieved. Typically, for M. tuberculosis complex the culture will be grown for approximately 3-4 weeks, and the optical density of the culture at 600 nm will be in the range of about 1.0 to 1.5, and more preferably will be in the range of about 1.0 to 1.2. Alternatively, if SPSN is to be utilized, the culture is grown for about 1-2 months prior to obtaining the supernatant. The supernatant can then be isolated and sterilized as described above, and utilized to revive or resuscitate dormant bacilli.
By xe2x80x9crevive or resuscitate dormant mycobacterium bacillixe2x80x9d and xe2x80x9cenhance growthxe2x80x9d we mean that a significant increase in the growth of a sample containing dormant bacilli is observed when the sample is cultured in the media of the present invention, compared to the growth of an equivalent sample cultured in conventional media. An equivalent sample would be one in which an equal amount of inoculum was introduced into an equal volume of media, and in which all other variables other than the presence/absence of isolated supernatant or components thereof, (e.g. temperature, degree of aeration, time of culturing, and the like) are held constant. By xe2x80x9cincrease in growthxe2x80x9d we mean an increase in the total number of bacteria in the culture, as determined by any of several techniques that are well-known to those of skill in the art. Such techniques include ascertaining the number of colony forming units (CFUs) present in the culture after incubation for a fixed amount of time, or by ascertaining the number of bacteria which test live in an FDA-EB test. In a preferred embodiment of the present invention, the quantitation of growth in the conventional and the supplemented medium may be carried out after a suitable time. For example, the cultures should be incubated for a minimum of about 2 days and for a maximum of about 7 to 28 days. The determination of growth in a culture may be carried out by any of a variety of techniques that are well-known to those of skill in the art, including but not limited to plating on solid media and visually observing colony formation (i.e. determining CFUs), observing an increase in turbidity in liquid culture as measured by optical density at absorption A600, or by utilizing a viability assay with redox dye such as MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium), or by detecting an increase in growth index (GI) value due to production of C14xe2x80x94CO2 in the Bactec TB460 system, and the like. The increase in growth that is observed, in order to be considered xe2x80x9csignificantxe2x80x9d is preferably at least about a two fold increase in growth. Alternatively, what may be observed is the emergence of any growth at all compared to a control in which no growth is observed without the resuscitation factors. In yet another embodiment of the present invention, a media is provided which is prepared from ESPSN and/or SPSN combined with fresh, conventional media. Examples of suitable media include but are not limited to 7H9, 7H10, 7H11, 7H12B, Sauton""s medium, Dubos medium, egg-based media such as Lowenstein-Jensen medium, and the like, which are readily commercially available (e.g. Difco). Further, those of skill in the art will recognize that such media may be enriched with suitable substances such as Tween 80, albumin-dextrose-catalase (ADC), fatty acids, and the like. The medium of the present invention may be prepared by combining ESPSN and/or SPSN with any suitable media, so long as the resulting medium appropriately sustains the growth of mycobacterium and supports the revival or resuscitation of dormant bacilli. Generally, the ratio of ESPSN and/or SPSN to media will be in the range of from about 0.1:1 to about 1:10, and more preferably in the range of from about 1:1 to about 1:2. Further, the media may also be further supplemented by the addition of substantially purified components of ESPSN and/or SPSN such as the proteins, peptides and phopholipids (or mixtures thereof) described below.
In another embodiment, the present invention further provides media for culturing mycobacterium which is comprised of a suitable culture medium supplemented with cell extract at least one substantially purified product of M. tuberculosis complex, the product exhibiting growth enhancing/resuscitation activity for dormant bacilli of the mycobacterium. Such products may be obtained from, for example, the ESPSN or the SPSN of a M. tuberculosis complex culture, or from cellular extracts of such a culture. While the examples recited herein disclose products from the ESPSN or the SPSN, those of skill in the art will recognize that those products are of cellular origin and are released into the supernatant from the bacteria. Thus, the products may also be obtained directly from cell extracts prior to their release from the cell. Alternatively, crude cell extracts may also be utilized. Methods of obtaining such extracts are well known to those of skill in the art and include but are not limited to sonication, French press, grinding with mortar and pestle, and the like. The amount of cell extract that can be added to fresh media to fabricate the supplemented media is in the range of about from 1:10 to about 1:1000, cell extract: media.
By xe2x80x9csubstantially purified productxe2x80x9d we mean a product which has been purified to contain no more than about 0-20%, and more preferably about 0-10%, and even more preferably about 0-5% extraneous material. Those of skill in the art will recognize that substantially purified components may contain trace amounts of material such as salts, ions (e.g. metal ions), and various other extraneous materials that do not interfere with the resuscitation activity exhibited by the substantially purified components.
In a preferred embodiment of the present invention, the mycobacterium is M. tuberculosis and the component of ESPSN and/or SPSN or cell extract is the protein Rv1147c or a peptide fragment of protein Rv1147c. By xe2x80x9cprotein Rv1147cxe2x80x9d we mean the protein corresponding to the amino acid sequence encoded by the open reading frame of the M. tuberculosis genome sequence which has been designated M. tuberculosis Rv1147c (accession number F70875 in the National Institute of Health""s Entrez Protein Database).
Those of skill in the art will recognize that other amino acid sequences which are not absolutely identical to the sequence of protein Rv1147c may also be utilized in the practice of the present invention. For example, proteins with various amino acid substitutions, or with various deletions or insertions in the sequence (e.g. such as those that may occur in variants of M. tuberculosis or which are generated via genetic engineering, etc.), or with various chemical modifications (e.g. acylation of the carboxy terminus), may also be utilized, so long as they retain the ability to function in the practice of the present invention. In general, such Rv1147c-based proteins will possess high homology to Rv1147c, i.e. in the range of about 75 to 100% homology, or more preferably in the range of about 85 to 100% homology, and most preferably in the range of about 95-100% homology to Rv1147c. Further, the protein Rv1147c may be from any source (e.g. isolated from M. tuberculosis, or from another organism into which the gene encoding the protein has been cloned, or fabricated synthetically, etc.)
Further, the component of ESPSN and/or SPSN or cell extract may be a peptide fragment of Rv1147c. Examples of such peptide fragments include but are not limited to those with the amino acid sequences of SEQ IDS NOS. 1-5 of the instant invention. However, those of skill in the art will recognize that many variations of these peptides may be made (for example, by varying the primary sequence of the peptides by amino acid substitutions, deletions or insertions, or by extending or shortening their length, etc.) and all such modified peptide fragments of Rv1147c are intended to be encompassed in the practice of the present invention. Any peptide fragment of Rv1147c may be utilized in the practice of the present invention, including peptides which are based on or are obvious variants of SEQ IDS 1-5. In general, the length of such a peptide fragment of Rv1147c will be from about 5 to about 20 amino acids. Further, the peptide fragments may be from any suitable source, e.g. they may be generated by chemical or proteolytic cleavage of Rv1147c or related proteins, they may be produced synthetically, or they may be produced via genetic engineering techniques. The particular source of the ESPSN and SPSN-based protein or peptides of the present invention is not a crucial feature of the invention.
The concentration of protein or peptide to be present in the media of the present invention may vary depending on the resuscitation activity of a given protein or peptide. However, it will generally be in about the picomolar to micromolar range. Typically, the concentration should be adequate to provide a significant increase in the CFU forming ability of dormant bacilli, compared to conventional, unsupplemented media.
In another embodiment of the present invention, the substantially purified component of ESPSN and/or SPSN or cell extract is a phospholipid or a component of a phospholipid. Phospholipids (also called phosphoglycerides) are composed of glycerol, phosphate and two fatty acyl units. Thus fatty acid components can have a carbon chain length of, for example, C18 (octadecanoic acid), C19 (nonadecanoic acid), 20 (eicosanoic acid), 21 (heneicosanoic acid), 22 (docosanoic acid), 23 (tricosanoic acid) 24 ((tetracosannoic acid), 25 (pentacosanoic acid), 26 (hexacosanoic acid), 27 (heptacosanoic acid), 28 (octacosanoic acid), 29 (nonacosanoic acid), 30 (triacontanoic acid), 31 (hentriacontanoic acid). Derivatives of these fatty acids, such as with double bonds and esters of these fatty acids can, either alone or in combination, be added to the culture media (7H12B, 7H9, 7H10, 7H11, Sauton""s medium, Dubos medium and egg-based media such as Lowenstein-Jensen medium) for improved diagnosis and treatment of mycobacterial infections. In a preferred embodiment of the present invention, the phospholipid or component of a phospholipid is phosphotidyl-L-serine, dioleoyl phosphotidyl-L-serine, phosphotidylcholine, phosphotidylethanolamine, tuberculostearic acid, arachidonic acid, C18-C31 fatty acids with or without double bonds, and esters of C18-C31 fatty acids with or without double bonds. Such phospholipids are well-known to those of skill in the art and are readily available.
The concentration of phospholipid or component thereof to be present in the media of the present invention may vary depending on the resuscitation activity of a given phospholipid. However, it will generally be in about the picomolar to micromolar range. Typically, the concentration should be adequate to provide a significant increase in the CFU forming ability of dormant bacilli, compared to conventional, unsupplemented media.
In addition, the media of the present invention may comprise more than one substantially purified component of ESPSN and/or SPSN or cell extract. For example, a combination of several peptide fragments, or of several lipids, or of several peptides and several lipids may also be utilized. In this case, the concentration of each component will typically be in the picomolar to micromolar range.
Types of media that may be supplemented by components of ESPSN and/or SPSN or cell extract include but are not limited to 7H12B, 7H9, 7H10, 7H11, Sauton""s medium, Dubos medium and egg-based media such as Lowenstein-Jensen medium. Further, those of skill in the art will recognize that the media of the present invention may be provided in any of several suitable forms. For example, the media may be provided in a premixed form (i.e. the components have already been added) or the components may be provided separately for addition to conventional media. Further, the media may be liquid (ready to use or concentrated) or solid.
The invention further provides methods for reviving dormant bacilli of a mycobacterium species. In one embodiment of the present invention, the method involves exposing dormant bacilli to isolated ESPSN or SPSN from M. tuberculosis complex cultures. The isolated ESPSN and SPSN will preferably have been sterilized, e.g. by filter sterilization.
In another embodiment of the present invention, the revival method involves exposing dormant bacilli of a mycobacterium species to a cell extract or at least one substantially purified product of M. tuberculosis complex (e.g. from the ESPSN, the SPSN, or a cell extract), the product exhibiting resuscitation activity for dormant bacilli of mycobacterium. In one embodiment, the product is a phospholipid or component thereof. In a preferred embodiment of the present invention, the phospholipid is phosphotidyl-L-serine, dioleoyl phosphotidyl-L-serine, phosphotidylcholine, phosphotidylethanolamine, tuberculostearic acid, arachidonic acid, or a fatty acid. In another embodiment, the product is a protein. In a preferred embodiment of the present invention the protein is Rv1147c or a peptide fragment of protein Rv1147c. Exemplary peptide fragments of Rv1147c include the peptides corresponding to SEQ ID NOS. 1-5.
Methods for the diagnosis of infections caused by a mycobacterium species (such as Mycobacterium tuberculosis, Mycobacterium paratuberculosis, and Mycobacterium leprae) are also contemplated. Improved methods are needed because, in the case of, for example, tuberculosis (caused by an M. tuberculosis infection), clinical specimens from suspected TB patients, especially those from patients under treatment, are known to contain injured and dormant bacilli, which may not grow in conventional culture media. In addition, bacilli in the specimens will also suffer from significant stress and injury during the processing of the clinical specimens by strong alkali NaOH and the centrifugation heat. Addition of the growth enhancing/resuscitation factors is likely to resuscitate the dormant or injured bacilli in the clinical specimens, resulting in a better recovery rate or improved sensitivity of primary isolation or the bacilli. The diagnostic methods of the instant invention are geared to detecting live and/or dormant M. tuberculosis bacilli with a far greater level of confidence. The diagnostic methods of the present invention may be used, for example, to screen persons suspected of having tuberculosis, or to monitor the progress of eradication of M. tuberculosis bacilli during or after treatment. In whatever context the diagnostic method is used, it would be highly advantageous to have the ability to detect dormant or injured forms of bacilli.
In one embodiment, the diagnostic method involves combining a sample for which the presence or absence of a mycobacterium species is to be ascertained with isolated early stationary phase supernatant of M. tuberculosis complex. The isolated ESPSN may have been sterilized e.g. by filter sterilization.
In another embodiment of the present invention, the diagnostic method involves combining a sample for which the presence or absence of a mycobacterium species is to be ascertained with a cell extract or at least one substantially purified product of M. tuberculosis complex (e.g. a component of ESPSN and/or SPSN culture supernatant or of a cell extract of the mycobacterium species). The cell extract or product exhibit the property of enhancing the growth of or resuscitating dormant bacilli of mycobacteria. In one embodiment, the product is a phospholipid or component of a phospholipid. In a preferred embodiment, the phospholipid or component thereof is, for example, phosphotidyl-L-serine, dioleoyl phosphotidyl-L-serine, phosphotidylcholine, phosphotidylethanolamine, tuberculostearic acid, arachidonic acid, or a fatty acid. In another embodiment, the product is the protein Rv1147c or a peptide fragment of protein Rv1147c. Exemplary peptide fragments of Rv1147c include the peptides corresponding to SEQ ID NOS. 1-5. Further, the method may also utilize combinations of such products.
Suitable samples for analysis by the methods of the present invention include but are not limited to sputum, blood, tissue, and the like.
The present invention also provides a kit for use in the diagnosis of infections caused by a mycobacterium species (such as Mycobacterium tuberculosis, Mycobacterium paratuberculosis, and Mycobacterium leprae. In one embodiment, the kit comprises a sealed container comprising isolated ESPSN and/or SPSN of M. tuberculosis complex. The isolated supernatant may have been sterilized e.g. by filter sterilization. In another embodiment of the invention, the kit comprises a sealed container comprising cell extract or at least one substantially purified product of M. tuberculosis complex, the product displaying the property of resuscitating dormant bacilli of mycobacteria, or generally enhancing the growth of the mycobacteria. In preferred embodiments, the product is a component of ESPSN, SPSN, or a cell extract of M. tuberculosis complex. In one embodiment, the component is a phospholipid or component of a phospholipid. In a preferred embodiment, the phospholipid or component thereof is, for example, phosphotidyl-L-serine, dioleoyl phosphotidyl-L-serine, phosphotidylcholine, phosphotidylethanolamine, tuberculostearic acid, arachidonic acid, or a fatty acid. In another embodiment, the product is a protein. In a preferred embodiment of the present invention, the protein Rv1147c or a peptide fragment of protein Rv1147c. Exemplary peptide fragments of Rv1147c include the peptides corresponding to SEQ ID NOS. 1-5. Further, the kit may also comprise combinations of such products, as well as such items as instructions for use of the kit.
Another aspect of the present invention is the treatment of infections caused by a mycobacterium species (such as Mycobacterium tuberculosis, Mycobacterium paratuberculosis, and Mycobacterium leprae). The treatment method involves the administration to a patient cell extract or at least one substantially purified product of M. tuberculosis complex, the product or extract displaying the property of resuscitating dormant bacilli of mycobacteria, or generally enhancing the growth of mycobacteria. In preferred embodiments, the product is a component of ESPSN, SPSN, or a cell extract of M. tuberculosis complex. In one embodiment, the component is a phospholipid or component of a phospholipid. In a preferred embodiment, the phospholipid or component is, for example, phosphotidyl-L-serine, dioleoyl phosphotidyl-L-serine, phosphotidylcholine, phosphotidylethanolamine, tuberculostearic acid, arachidonic acid, or a fatty acid. In another embodiment, the product is a protein. In a preferred embodiment of the present invention, the protein is Rv1147c or a peptide fragment of protein Rv1147c. Exemplary peptide fragments of Rv1147c include the peptides corresponding to SEQ ID NOS. 1-5. The components is administered in conjunction with an established drug treatment regimen such as DOTS. The administration of such components serves to revive dormant bacilli in the patient, making them susceptible to eradication by drug therapy. Otherwise, only non-dormant bacilli are eliminated and the patient is at risk for relapsing due to revival of the dormant bacilli after the treatment regimen has ceased. A combination of the above (e.g. more than one phospholipid, more than one peptide, or a combination of lipids and peptides) may also be administered. The quantity of the component to be administered may vary from patient to patient, depending on factors such as weight, gender, age, and general health, etc. of the patient, and it best determined by a skilled practitioner such as a physician. Generally, however, when a phospholipid is administered, the dosage will be in about the micromolar range, and when a protein or peptide is administered, the dosage will be in about the micromolar range.
Administration may be effected by any of a variety of routes that are well-known to those of skill in the art, including but not limited to oral, parenteral, intravenous, via inhalation, and the like.
To that end, the invention also provides a pharmacological preparation comprising cell extract or at least one substantially purified product of M. tuberculosis complex, the product displaying the property of resuscitating dormant bacilli of mycobacteria, or generally enhancing the growth of mycobacterium. The component may be a phospholipid or a component of a phospholipid, or a protein or fragment of the protein. In a preferred embodiment of the present invention, the phospholipid or component of a phospholipid is, for example, phosphotidyl-L-serine, dioleoyl phosphotidyl-L-serine, phosphotidylcholine, phosphotidylethanolamine, tuberculostearic acid, arachidonic acid, or a fatty acid. In another embodiment, the component is the protein Rv1147c or a peptide fragment of protein Rv1147c. Exemplary peptide fragments of Rv1147c include the peptides corresponding to SEQ ID NOS. 1-5. The pharmalogical preparation may also comprise a combination of the above (e.g. more than one phospholipid, more than one peptide, or a combination of lipids and peptides. Such a pharmacological preparation may also comprise other suitable substances and excipients, including but not limited to physiological acceptable buffering agents, stabilizers (e.g. antioxidants), flavoring agents, and the like. Further, the pharmacological preparation may be in any of a variety of art-accepted forms such as tablets, capsules, various injectable formulations, and the like, as are suitable for the desired means of administration.
In yet another aspect, the present invention provides a method of inhibiting the growth of a mycobacterium species such as Mycobacterium tuberculosis, Mycobacterium paratuberculosis, or Mycobacterium leprae. The method involves exposing the mycobacterium species to the isolated and sterilized supernatant of a culture M. tuberculosis complex or a cell extract of M. tuberculosis complex that is at least about 3 months in age. Alternatively, the method involves exposure of the mycobacterium species to substantially purified products from such a culture, wherein the products exhibit the property of inhibiting the growth of the mycobacterium species.